Abstract:

It is an object of the present invention to provide antibodies which
recognize non-reducing mannose residues. The present invention provides
antibodies recognizing non-reducing mannose residues, which are obtained
through screening of phage-display library of human scFvs by using, as an
antigen, Man3-DPPE which is an artificial glycolipid synthesized from
mannotriose and dipalmitoylphosphatidylethanolamine by reductive
amination.

Claims:

1. An antibody of any of the following (1) to (14):(1) an antibody
recognizing a non-reducing mannose residue, comprisingthe amino acid
sequence of SEQ ID NO: 2 or an amino acid sequence, wherein one or a few
amino acids have been deleted, substituted, or added in the amino acid
sequence of SEQ ID NO: 2, as an amino acid sequence of heavy chain
variable region, and the amino acid sequence of SEQ ID NO: 4 or an amino
acid sequence, wherein one or a few amino acids have been deleted,
substituted, or added in the amino acid sequence of SEQ ID NO: 4, as an
amino acid sequence of light chain variable region;(2) an antibody
recognizing a non-reducing mannose residue, comprisingthe amino acid
sequence of SEQ ID NO: 6 or an amino acid sequence, wherein one or a few
amino acids have been deleted, substituted, or added in the amino acid
sequence of SEQ ID NO: 6, as an amino acid sequence of heavy chain
variable region, and the amino acid sequence of SEQ ID NO: 8 or an amino
acid sequence, wherein one or a few amino acids have been deleted,
substituted, or added in the amino acid sequence of SEQ ID NO: 8, as an
amino acid sequence of light chain variable region;(3) an antibody
recognizing a non-reducing mannose residue, comprisingthe amino acid
sequence of SEQ ID NO: 10 or an amino acid sequence, wherein one or a few
amino acids have been deleted, substituted, or added in the amino acid
sequence of SEQ ID NO: 10, as an amino acid sequence of heavy chain
variable region, and the amino acid sequence of SEQ ID NO: 12 or an amino
acid sequence, wherein one or a few amino acids have been deleted,
substituted, or added in the amino acid sequence of SEQ ID NO: 12, as an
amino acid sequence of light chain variable region;(4) an antibody
recognizing a non-reducing mannose residue, comprisingthe amino acid
sequence of SEQ ID NO: 14 or an amino acid sequence, wherein one or a few
amino acids have been deleted, substituted, or added in the amino acid
sequence of SEQ ID NO: 14, as an amino acid sequence of heavy chain
variable region, and the amino acid sequence of SEQ ID NO: 16 or an amino
acid sequence, wherein one or a few amino acids have been deleted,
substituted, or added in the amino acid sequence of SEQ ID NO: 16, as an
amino acid sequence of light chain variable region;(5) an antibody
recognizing a non-reducing mannose residue, comprisingthe amino acid
sequence of SEQ ID NO: 18 or an amino acid sequence, wherein one or a few
amino acids have been deleted, substituted, or added in the amino acid
sequence of SEQ ID NO: 18, as an amino acid sequence of heavy chain
variable region, and the amino acid sequence of SEQ ID NO: 20 or an amino
acid sequence, wherein one or a few amino acids have been deleted,
substituted, or added in the amino acid sequence of SEQ ID NO: 20, as an
amino acid sequence of light chain variable region;(6) an antibody
recognizing a non-reducing mannose residue, comprisingthe amino acid
sequence of SEQ ID NO: 22 or an amino acid sequence, wherein one or a few
amino acids have been deleted, substituted, or added in the amino acid
sequence of SEQ ID NO: 22, as an amino acid sequence of heavy chain
variable region, and the amino acid sequence of SEQ ID NO: 24 or an amino
acid sequence, wherein one or a few amino acids have been deleted,
substituted, or added in the amino acid sequence of SEQ ID NO: 24, as an
amino acid sequence of light chain variable region;(7) an antibody
recognizing a non-reducing mannose residue, comprisingthe amino acid
sequence of SEQ ID NO: 26 or an amino acid sequence, wherein one or a few
amino acids have been deleted, substituted, or added in the amino acid
sequence of SEQ ID NO: 26, as an amino acid sequence of heavy chain
variable region, and the amino acid sequence of SEQ ID NO: 28 or an amino
acid sequence, wherein one or a few amino acids have been deleted,
substituted, or added in the amino acid sequence of SEQ ID NO: 28, as an
amino acid sequence of light chain variable region;(8) an antibody
recognizing a non-reducing mannose residue, comprisingthe amino acid
sequence of SEQ ID NO: 30 or an amino acid sequence, wherein one or a few
amino acids have been deleted, substituted, or added in the amino acid
sequence of SEQ ID NO: 30, as an amino acid sequence of heavy chain
variable region, and the amino acid sequence of SEQ ID NO: 32 or an amino
acid sequence, wherein one or a few amino acids have been deleted,
substituted, or added in the amino acid sequence of SEQ ID NO: 32, as an
amino acid sequence of light chain variable region;(9) an antibody
recognizing a non-reducing mannose residue, comprisingthe amino acid
sequence of SEQ ID NO: 34 or an amino acid sequence, wherein one or a few
amino acids have been deleted, substituted, or added in the amino acid
sequence of SEQ ID NO: 34, as an amino acid sequence of heavy chain
variable region, and the amino acid sequence of SEQ ID NO: 36 or an amino
acid sequence, wherein one or a few amino acids have been deleted,
substituted, or added in the amino acid sequence of SEQ ID NO: 36, as an
amino acid sequence of light chain variable region;(10) an antibody
recognizing a non-reducing mannose residue, comprisingthe amino acid
sequence of SEQ ID NO: 38 or an amino acid sequence, wherein one or a few
amino acids have been deleted, substituted, or added in the amino acid
sequence of SEQ ID NO: 38, as an amino acid sequence of heavy chain
variable region, and the amino acid sequence of SEQ ID NO: 40 or an amino
acid sequence, wherein one or a few amino acids have been deleted,
substituted, or added in the amino acid sequence of SEQ ID: 40, as an
amino acid sequence of light chain variable region;(11) an antibody
recognizing a non-reducing mannose residue, comprisingthe amino acid
sequence of SEQ ID NO: 42 or an amino acid sequence, wherein one or a few
amino acids have been deleted, substituted, or added in the amino acid
sequence of SEQ ID NO: 42, as an amino acid sequence of heavy chain
variable region, and the amino acid sequence of SEQ ID NO: 44 or an amino
acid sequence, wherein one or a few amino acids have been deleted,
substituted, or added in the amino acid sequence of SEQ ID: 44, as an
amino acid sequence of light chain variable region; and(12) an antibody
recognizing a non-reducing mannose residue, comprisingthe amino acid
sequence of SEQ ID NO: 46 or an amino acid sequence, wherein one or a few
amino acids have been deleted, substituted, or added in the amino acid
sequence of SEQ ID NO: 46, as an amino acid sequence of heavy chain
variable region, and the amino acid sequence of SEQ ID NO: 48 or an amino
acid sequence, wherein one or a few amino acids have been deleted,
substituted, or added in the amino acid sequence of SEQ ID NO: 48, as an
amino acid sequence of light chain variable region.

[0017]the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence,
wherein one or a few amino acids have been deleted, substituted, or added
in the amino acid sequence of SEQ ID NO: 2, as an amino acid sequence of
heavy chain variable region, and the amino acid sequence of SEQ ID NO: 4
or an amino acid sequence, wherein one or a few amino acids have been
deleted, substituted, or added in the amino acid sequence of SEQ ID NO:
4, as an amino acid sequence of light chain variable region;

[0018]the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence,
wherein one or a few amino acids have been deleted, substituted, or added
in the amino acid sequence of SEQ ID NO: 6, as an amino acid sequence of
heavy chain variable region, and the amino acid sequence of SEQ ID NO: 8
or an amino acid sequence, wherein one or a few amino acids have been
deleted, substituted, or added in the amino acid sequence of SEQ ID NO:
8, as an amino acid sequence of light chain variable region;

[0019]the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence,
wherein one or a few amino acids have been deleted, substituted, or added
in the amino acid sequence of SEQ ID NO: 10, as an amino acid sequence of
heavy chain variable region, and the amino acid sequence of SEQ ID NO: 12
or an amino acid sequence, wherein one or a few amino acids have been
deleted, substituted, or added in the amino acid sequence of SEQ ID NO:
12, as an amino acid sequence of light chain variable region;

[0020]the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence,
wherein one or a few amino acids have been deleted, substituted, or added
in the amino acid sequence of SEQ ID NO: 14, as an amino acid sequence of
heavy chain variable region, and the amino acid sequence of SEQ ID NO: 16
or an amino acid sequence, wherein one or a few amino acids have been
deleted, substituted, or added in the amino acid sequence of SEQ ID NO:
16, as an amino acid sequence of light chain variable region;

[0021]the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence,
wherein one or a few amino acids have been deleted, substituted, or added
in the amino acid sequence of SEQ ID NO: 18, as an amino acid sequence of
heavy chain variable region, and the amino acid sequence of SEQ ID NO: 20
or an amino acid sequence, wherein one or a few amino acids have been
deleted, substituted, or added in the amino acid sequence of SEQ ID NO:
20, as an amino acid sequence of light chain variable region;

[0022]the amino acid sequence of SEQ ID NO: 22 or an amino acid sequence,
wherein one or a few amino acids have been deleted, substituted, or added
in the amino acid sequence of SEQ ID NO: 22, as an amino acid sequence of
heavy chain variable region, and the amino acid sequence of SEQ ID NO: 24
or an amino acid sequence, wherein one or a few amino acids have been
deleted, substituted, or added in the amino acid sequence of SEQ ID NO:
24, as an amino acid sequence of light chain variable region;

[0023]the amino acid sequence of SEQ ID NO: 26 or an amino acid sequence,
wherein one or a few amino acids have been deleted, substituted, or added
in the amino acid sequence of SEQ ID NO: 26, as an amino acid sequence of
heavy chain variable region, and the amino acid sequence of SEQ ID NO: 28
or an amino acid sequence, wherein one or a few amino acids have been
deleted, substituted, or added in the amino acid sequence of SEQ ID NO:
28, as an amino acid sequence of light chain variable region;

[0024]the amino acid sequence of SEQ ID NO: 30 or an amino acid sequence,
wherein one or a few amino acids have been deleted, substituted, or added
in the amino acid sequence of SEQ ID NO: 30, as an amino acid sequence of
heavy chain variable region, and the amino acid sequence of SEQ ID NO: 32
or an amino acid sequence, wherein one or a few amino acids have been
deleted, substituted, or added in the amino acid sequence of SEQ ID NO:
32, as an amino acid sequence of light chain variable region;

[0025]the amino acid sequence of SEQ ID NO: 34 or an amino acid sequence,
wherein one or a few amino acids have been deleted, substituted, or added
in the amino acid sequence of SEQ ID NO: 34, as an amino acid sequence of
heavy chain variable region, and the amino acid sequence of SEQ ID NO: 36
or an amino acid sequence, wherein one or a few amino acids have been
deleted, substituted, or added in the amino acid sequence of SEQ ID NO:
36, as an amino acid sequence of light chain variable region;

[0026]the amino acid sequence of SEQ ID NO: 38 or an amino acid sequence,
wherein one or a few amino acids have been deleted, substituted, or added
in the amino acid sequence of SEQ ID NO: 38, as an amino acid sequence of
heavy chain variable region, and the amino acid sequence of SEQ ID NO: 40
or an amino acid sequence, wherein one or a few amino acids have been
deleted, substituted, or added in the amino acid sequence of SEQ ID: 40,
as an amino acid sequence of light chain variable region;

[0027]the amino acid sequence of SEQ ID NO: 42 or an amino acid sequence,
wherein one or a few amino acids have been deleted, substituted, or added
in the amino acid sequence of SEQ ID NO: 42, as an amino acid sequence of
heavy chain variable region, and the amino acid sequence of SEQ ID NO: 44
or an amino acid sequence, wherein one or a few amino acids have been
deleted, substituted, or added in the amino acid sequence of SEQ ID: 44,
as an amino acid sequence of light chain variable region; and

[0028]the amino acid sequence of SEQ ID NO: 46 or an amino acid sequence,
wherein one or a few amino acids have been deleted, substituted, or added
in the amino acid sequence of SEQ ID NO: 46, as an amino acid sequence of
heavy chain variable region, and the amino acid sequence of SEQ ID NO: 48
or an amino acid sequence, wherein one or a few amino acids have been
deleted, substituted, or added in the amino acid sequence of SEQ ID NO:
48, as an amino acid sequence of light chain variable region.

[0029]Another aspect of the present invention provides a nucleic acid
encoding the aforementioned antibody of the present invention.

[0043]Further another aspect of the present invention provides a process
for producing an antibody wherein the aforementioned nucleic acid of the
present invention is used.

[0044]Further another aspect of the present invention provides a
recombinant antibody recognizing a nonreducing mannose residue which can
be obtained by the aforementioned process. Preferably, the antibody is a
single chain antibody.

BEST MODE FOR CARRYING OUT THE INVENTION

[0045]Hereafter, embodiments of the present invention will be more
specifically described.

[0046]The antibodies of the present invention are antibodies which
recognize non-reducing mannose residues. As described in the Examples
below, the antibodies of the present invention have been obtained through
screening of phage-display library of human scFvs, with use of Man3-DPPE,
as a model antigen, which is an artificial glycolipid synthesized from
mannotriose (Man3) and dipalmitoylphosphatidylethanolamine (DPPE) by
reductive amination. As a result of characterization of positive phage
clones by TLC-overlay assays and surface plasmon resonance analyses, it
was demonstrated that the antibodies of the present invention bound to
artificial glycolipids bearing mannose residues at nonreducing termini.

[0047]The glycolipid for use as an antigen in screening of the present
invention is not specifically limited, and any artificial glycolipid
bearing a mannose residue at a nonreducing terminal may be used, although
oligosaccharides are preferred.

[0048]In an oligosaccharide, individual constituent sugars bind to one
another via an α→1,2 bond, an α→1,3 bond, an
α→1,4 bond, an α→1,6 bond, β→1,4
bond, or a combination thereof. For example, mannose may constitute a
straight chain through the abovementioned bond, or may adopt a branched
structure via the combination of an α→1,3 bond with an
α→1,6 bond. The number of monosaccharides in an
oligosaccharide is preferably 2 to 11.

[0050]These oligosaccharides have a reducing terminal aldehyde group.
Thus, such an aldehyde group can be used as a means for immobilizing an
oligosaccharide. That is to say, an aldehyde group is allowed to react
with a lipid having an amino group to form a Schiff base. Subsequently,
according to a common method, the Schiff base is reduced, and is
preferably chemically reduced by NaBH3CN, for example, so as to bind
the oligosaccharide to the lipid.

[0051]The aforementioned lipid having an amino group is preferably a
phospholipid having an amino group. For example, phosphatidylamine such
as dipalmitoylphosphatidylethanolamine (DPPE) or
distearoylphosphatidylethanolamine (DSPE) can be used. Thus obtained
bound substance of an oligosaccharide and a lipid may be referred to as
an artificial glycolipid in the present invention. The glycolipid for use
in the present invention is preferably an artificial glycolipid as
mentioned above.

[0052]The solvent for dissolving an artificial glycolipid serving as an
antigen is not specifically limited, although it is preferable to
dissolve such an artificial glycolipid in a solvent system of
chloroform/methanol/water (10:10:3, v/v) and subsequently dilute it with
methanol at 40 μg/ml, for example. This solution is added in, for
example, a 96-well plastic plate (flat bottom) and dried in air so as to
immobilize the artificial glycolipid onto the wells. These wells are
blocked and washed. The plate coated with an artificial glycolipid by
such an operation can be used as an antigen-coated plate.

[0053]Tris-buffered saline (hereunder, referred to as TBS) is desirably
used as the buffer solution. In addition, for example, TBS containing 3%
bovine serum albumin (hereunder, referred to as 3% BSA/TBS) is desirable
as the blocking solution, and TBS containing 0.2% Tween 20 (hereunder,
referred to as TBS-T) is desirable as the washing solution.

[0055]The phage-display antibody library is constructed by adapting a
system in which antibodies are fused with the coat protein of a
filamentous phage to thereby display these antibodies on the phage
surface. Specifically, such a phage-display library can be made by PCR
amplification of antibody genes to produce a library including many types
of antibody genes, and displaying them on a phage. Hereunder, specific
example of the method for producing phage-displayed single chain
antibodies is described, but is not to be considered as limiting.

[0056]Human peripheral blood cDNA library and human spleen cDNA library
are used as templates to amplify a fragment including CDR1 and CDR2
regions in VH or VL region of immunoglobulin gene, and a fragment
including CDR3 region by PCR. These fragments are mixed and used as
templates to amplify the VH or VL region by PCR. These amplified DNA
fragments are respectively inserted into a non-expressing phagemid vector
to produce a VH library and a VL library. These E. coli VH library and VL
library are infected with helper phages to convert into phage libraries.
These phage-type VH library and VL library are coinfected into E. coli
expressing Cre-recombinase, to effect recombination between the VH and VL
vectors within E. coli. This E. coli strain is infected with helper
phages M13KO7, by which phages expressing full length single-chain
antibodies can be produced. To remove unrecombinants mixed in these
phages, E. coli is infected with these phages while avoiding multiple
infection, then superinfected with helper phages, and incubated in a
medium which contains ampicillin/kanamycin/chloramphenicol but does not
contain glucose at 25° C. By so doing, phages expressing
recombinant single-chain antibodies can be obtained in the supernatant.
The phages in the supernatant are precipitated with polyethylene glycol
and resuspended, by which the artificial antibody library with a large
repertoire (1011 or more clones) can be obtained.

[0057]Further, a phage-displayed antibody having a binding property to a
carbohydrate of interest can be selectively collected from the human
single-chain phage library produced by the above manner, with reference
to the binding property to an artificial glycolipid having the concerned
carbohydrate structure. This operation may be referred to as panning.

[0058]The carbohydrate-specific binding property of phage-displayed
antibodies obtained by repetition of panning is desirably analyzed by the
ELISA method that will be described later, for example, because a large
number of phage clone samples can be simultaneously analyzed. That is to
say, a carbohydrate is immobilized onto a 96-well plastic plate and
blocked with a buffer solution containing 3% BSA for example, in order to
avoid non-specific adsorption of phage-displayed antibodies. A suspension
of the phage-displayed antibodies which had been adjusted at an
appropriate concentration with a buffer solution is added thereto,
followed by sufficient reaction (for example, at 37° C. for 2
hours), and subsequent washing with a buffer solution.

[0059]To detect/quantify phage-displayed antibodies bound to the
carbohydrate, antibodies are reacted with an aqueous solution containing
anti-phage antibodies that have been labeled with a substance suitable
for detection, followed by washing with a buffer solution, and subsequent
detection/quantification in an optimum method for detecting the labeling
substance. At this time, there is no specific limitation in the labeling
substance for the anti-phage antibodies, although labeling with
horseradish peroxidase is desirable for example, in terms of handiness.
In addition, there is no specific limitation in the detector for use in
the detection, although a plate reader suitable for ELISA is desirably
employed.

[0060]The term "antibody" used in the present invention does not only
refer to antibodies in a form normally existing in vivo, but also include
peptides containing at least one antigen-binding site formed of the
variable region in the H chain or L chain of the antibody, or a
combination thereof, Fab composed of a set of an H chain fragment and an
L chain fragment, F(ab')2 composed of two sets of H chain fragments
and L chain fragments, single chain antibodies composed of an H chain
fragment and an L chain fragment bound in series in a single peptide
(hereunder, may be referred to as "scFvs"), and the like. The "antibody"
of the present invention may be a full length antibody in a form normally
existing in vivo, which is composed of two sets of full length H chains
and full length L chains.

[0061]The terms "F(ab')2" and "Fab" in the present invention mean
antibody fragments produced by treatment of an immunoglobulin with a
protease such as pepsin or papain, and generated by digestion around the
disulfide bonds existing between two H chains in the hinge region. For
example, when IgG1 is treated with papain, cleavages occur in upstreams
of the disulfide bonds existing between two H chains in the hinge region
to allow the production of two identical antibody fragments in which an L
chain composed of VL (L chain variable region) and CL (L chain constant
region) and an H chain fragment composed of VH (H chain variable region)
and CHγ1 (γ1 region within H chain constant region) are
connected by a disulfide bond at the C-terminal region. These two
identical antibody fragments are respectively referred to as Fab'. In
addition, when IgG is treated with pepsin, cleavages occur in downstreams
of the disulfide bonds existing between two H chains in the hinge region
to allow the production of an antibody fragment which is slightly larger
than the combined product having said two Fab's connected by the hinge
region. This antibody fragment is referred to as F(ab')2.

[0062]Usually, an antibody consists of two types of large and small
polypeptides. The large subunit is referred to as "H chain (heavy chain)"
and the small subunit is referred to as "L chain (light chain)". In
addition, each peptide is composed of a "variable region" existing at the
N-terminal side and forming an antigen-binding site, and a "constant
region" which is conserved per each antibody class. The variable region
is further divided into complementarity determining regions "CDRs" which
particularly involve the formation of the antigen-binding site, and
"framework regions" existing therebetween. CDRs are known consist of
three regions called "CDR1", "CDR2", and "CDR3" from the N-terminal side,
for each H chain and L chain.

[0063]The single chain antibodies of the present invention can be prepared
by appropriately selecting inducible vectors such as pSE380 plasmid
(Invitrogen) or pET24d(+) plasmid (Novagen) and host bacterial cells. In
addition to the above method, in the production of the antibodies of the
present invention, an animal cell expression system, an insect cell
expression system, and a yeast cell expression system can also be used.
The linker for linking the H chain and the L chain can also be
appropriately selected by those skilled in the art.

[0113]Further, an antibody in a form normally existing in vivo can be
prepared from scFv. For example, only the variable regions of the H chain
and the L chain are amplified by PCR from a scFv plasmid. Each fragment
is, for example, recombined into a plasmid having the H chain gene and/or
the L chain gene of a human antibody, which thereby enables the formation
of an antibody having a variable region on the scFv in a form normally
existing in vivo. Specifically, for example, appropriate restriction
enzyme cleavage sites are introduced at both ends of the gene fragment
obtained when amplifying the variable regions of the H chain and the L
chain from the plasmid, and they are combined with an appropriate
restriction enzyme cleavage site on the plasmid having the H chain and/or
the L chain of the human antibody, thereby replacing genes in the
variable region without causing frame-shift. Thus, an antibody in a form
normally existing in vivo which has a sequence of a variable region on a
plasmid as it is, can be prepared. Further, a peptide containing at least
one antigen-binding site formed of the variable region of the H chain or
L chain of the antibody, or a combination thereof, Fab composed of a set
of an H chain fragment and an L chain fragment, and (Fab'2) composed of
two sets of H chain fragments and L chain fragments can also be prepared
from the antibody.

[0114]The expression of the antibody of the present invention can be
carried out by employing E. coli, yeast, insect cells, animal cells, and
the like. For example, when an antibody is expressed in COS cell or CHO
cell, pCDNA3.1(+) or pMAMneo (CLONETECH) can be used. For example, a gene
of the H chain of the antibody obtained in the above method is
incorporated into a multicloning site of pCDNA3.1(+), and a gene of the L
chain is incorporated into pMAMneo. Then, an expression unit having a
gene of the L chain between a promoter and poly A is incorporated into an
adequate site of the vector having the H chain incorporated therein.
Introduction of this vector into a COS cell, a CHO-K1, or a CHO DG44 by a
conventional genetic engineering technique enables the production of the
antibody of interest. Further, the expression unit of the DHFR gene is
cleaved out from for example, pSV2/DHFR (Nature, 1981. Vol. 294, Lee F.
et al.) into the above prepared vector, and is incorporated into a vector
which expresses the H chain and the L chain. This vector is introduced
into the CHO DG44 by a conventional genetic engineering technique. Thus
selected cells can be used to significantly improve the productivity of
antibodies by utilizing the DHFR gene amplification system using MTX.

[0115]Animal cells such as COS cell or CHO cell can be generally cultured
in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal
bovine serum (FBS) under 5% CO2 at 37° C. A method for
introducing a gene into the COS cell may be electroporation, as well as a
DEAE-dextran method and a method using a transfection reagent such as
lipofectin.

[0116]At the time of production of the antibody of the present invention,
the cells are preferably cultured in a serum-free medium in order to
prevent the contamination of a serum-derived bovine antibody. COS and CHO
cells which are not acclimatized in a serum-free medium but are cultured
in serum media, are preferably cultured in serum-free DMEM. The antibody
of the present invention, which is thus obtained in the culture
supernatant, can be easily purified by a conventional method for
purifying IgG antibodies using, for example, Protein A column and Protein
G column.

[0121]PCR templates used in this study were human spleen cDNA purchased
from BioChain (Hayward, Calif.) and human leukocyte cDNA purchased from
BD Biosciences (Palo Alto, Calif.). The human immunoglobulin variable
regions were amplified by PCR using an equimolar mixture of appropriate
forward and reverse primers having sequences in common with this gene
family, according to previously published information (Sbalattero, D. &
Bradbury, A. (1998) Immunotechnology 3, 271-278).

[0122]Reaction mixtures (50 μl) contained 0.5 μl of the cDNA
solution, 25 pmol of each of forward primer and reverse primer, 200 μM
dNTPs, 10 mM KCl, 10 mM (NH4)2SO4, 20 mM Tris-HCl (pH
8.8), 2 mM MgCl2, 100 μg/ml BSA, and 1 unit of KOD plus DNA
polymerase (Toyobo, Osaka, Japan). The PCR condition was optimized for
each primer set. The first round PCR products purified by agarose gel
electrophoresis were mixed in the same quantity as used for templates in
the following PCR to amplify immunoglobulin variable regions. The
resulting DNA fragments of the variable regions were purified and
digested with appropriate restriction enzymes followed by insertion into
pVH-Hyg or pVL-Amp vectors. After ligation, the resulting phagemid DNA
was electroporated into E. coli DH10B strain harboring F' plasmid derived
from E. coli XL1-Blue, and colonies were grown at 30° C. on agar
plates containing 1% glucose and hygromycin (50 μg/ml) or ampicillin
(50 μg/ml). The colonies were collected and converted to phages
bearing VH or VL genes by superinfection with helper phage M13KO7 in
2× YT medium containing 1% glucose at 30° C. overnight. The
phages were collected by centrifugation and precipitation with
polyethylene glycol (PEG) according to a standard protocol.

Recombination and Generation of Secondary Phage scFv Library

[0123]The F' plasmid derived from E. coli XL1-Blue strain was introduced
into E. coli NS3529 strain, which expresses Cre-recombinase
constitutively, to obtain E. coli NS3529/F' strain. The NS3529/F' strain
was infected with VH and VL phages at MOI=50, and was left still at
37° C. for 1 hour without shaking, and was then superinfected with
M13KO7 helper phages at MOI=20. After samples were incubated at
37° C. for 1 hour, ampicillin and kanamycin were added at 50
μg/ml and 25 μg/ml, respectively. During overnight culture at
37° C., Cre/lox recombination resulted in formation of scFv genes
because of insertion of a VH fragment into the pVL-Amp vector in the
NS3529/F' cells. Growing XL1-Blue strain was infected with the
recombinant phages obtained from the culture at MOI<0.1, and was left
still at 37° C. for 1 hour. ScFv displaying phages fused with a
coat protein g3p were obtained from the strain which was cultured in
2× YT medium (glucose free) containing 20 μg/ml chloramphenicol,
50 μg/ml ampicillin and 25 μg/ml kanamycin at 25° C.
overnight. The phages which were concentrated by PEG precipitation were
suspended in SM buffer containing 1% gelatin and 6% DMSO, and were stored
at -80° C.

(4) Phage Selection Methods

Panning Procedures

[0124]The library was subjected to four rounds of panning. Fifty and
sixteen wells of a 96-well plate were coated with Man3-DPPE (2
μg/well) and used for first and second panning, respectively. Five and
two wells coated with Man3-DPPE (2 μg/well) were used for third and
fourth panning, respectively. Phage selection was basically carried out
according to previously published procedures (Marks, J. D., Hoogenboom,
H. R., Bonnet, T. P., McCafferty, J., Griffiths, A. D., and Winter, G.
(1991) J. Mol. Biol. 222, 581-597) with some modifications. Coating of
wells with Man3-DPPE is described briefly. Wells are applied with 50
μl of Man3-DPPE (40 μg/ml methanol solution) and the solvent is
dried at 37° C., followed by incubation with 150 μl of Tris-HCl
buffer (pH 7.4) containing 0.15 M NaCl and 3% BSA (blocking buffer) at
4° C. overnight. The wells were rinsed twice with 50 μl of 0.2%
Tween 20/TBS (TBS-T), and once with 200 μl of TBS. 50 μl of phage
suspension in TBS containing 0.1% Tween 20 and 1.4% BSA was added to the
wells, which were then incubated at 37° C. for 60 minutes under
shaking. After the wells were washed 3 times with 200 μl of TBS-T and
twice with 200 μl of TBS, bound phages were eluted by addition of 50
μl of 100 mM triethylamine and incubation at 25° C. for 10
minutes. Then, the resultant product was neutralized by mixing with 100
μl of neutralizing solution (1 M Tris-HCl (pH 7.4):3% BSA/TBS=2:1,
v/v) in wells of the control plate, which had been treated with the
blocking buffer. Bound phages were further eluted by addition of 50 μl
of 100 mM triethylamine and incubation at 25° C. for 20 minutes
and were recovered in the neutralization solution as described above.
Eluted phages were used to infect 100 μl of logarithmic growing E.
coli TG1 strain at 37° C. for 1 hour. Infected bacteria were grown
on LB agar plates (medium) (diameter=10 cm) containing 1 mM NaOH, 0.1%
glucose, and carbenicillin (50 μg/ml) at 25° C. for 16 hours,
and then were collected from the plates using a spreader after addition
of 2 ml of LB-10 mM Tris-HCl (pH 7.5) (SBS) per plate. 1 ml out of the 38
ml of this suspension was inoculated into 40 ml of SBS containing
carbenicillin and grown with shaking at 37° C. for 2 hours. 40
μl of solution containing 3.5×109 pfu. of helper phages was
added and the resultant was incubated at 37° C. for 1 hour without
shaking. Then, kanamycin (25 μg/ml)/chloramphenicol (10 μg/ml) was
added, and the resultant was incubated with rotation at 25° C. for
40 hours. Thus, phages were collected. Phage particles were concentrated
by PEG-precipitation, and were dissolved in 400 μl of TBS, 400 μl
of 3% BSA/TBS, and 40 ml of 10% Tween 20/TBS by incubation at 37°
C. for 1 hour. After centrifugation (at 18,000 g at 4° C. for 5
minutes), 800 μl of phage suspension were recovered and used for
second panning. Second, third, and fourth panning were carried out
similarly to first panning except for washing conditions. Bacteria picked
from single colonies after the fourth panning were grown in 50 μl of
SBS/carbenicillin in 96 well plates at 37° C. for 1 hour with
rotation, to which 50 μl of helper phages were added and incubated at
37° C. for 1 hour with rotation. After
SBS/kanamycin/chloramphenicol mixture was added at 50 μl/well, phage
suspensions were collected through incubation at 25° C. for 16
hours with rotation and centrifugation (at 200 g at 4° C. for 15
minutes). 50 μl of the supernatants were added to wells containing 100
μl of 3% BSA/TBS, incubated at 37° C. for 1 hour, and kept at
4° C.

[0125]Analysis of binding of phages to Man3-DPPE was performed by ELISA
using bacterial supernatants containing phages. A 96-well plate was
coated with Man3-DPPE (1 μg/well) as described above and blocked by
incubation with 150 μl of 3% BSA/TBS at 4° C. overnight.
Control plates were prepared as above without antigen. 75 μl of phage
suspensions were added to the wells and incubated at 37° C. for 1
hour. The wells were washed 5 times with 200 μl of TBS-T. Bound phage
antibodies were detected by incubation with horseradish peroxidase
(HRP)-labeled anti-M13 antibody at 37° C. for 1 hour, after which
they were washed 10 times with 200 μl of TBS-T and once with 200 μl
of TBS. Peroxidase activity was detected by reaction with
ABTS/H2O2 for 30 minutes and termination with 1% oxalic acid.
Then, the absorbance was measured with a BIO-RAD plate-reader at 415 nm.

(5) Characterization of Phage Antibodies

Colony PCR and Determination of DNA Sequences

[0126]scFv genes were amplified from respective E. coli TG1 colonies
infected with phages by PCR with a primer set (forward primer Cm-f:
5'-TGTGATGGCTTCCATGTCGGCAGAATGCT-3' (SEQ ID NO: 97), and reverse primer
g3-r: 5'-GCTAAACAACTTTCAACAGTCTATGCGGCAC-3' (SEQ ID NO: 98)). After
preheating the sample at 94° C. for 2 minutes, PCR was carried out
with 35 cycles under conditions of denaturing at 94° C. for 20
seconds, annealing at 53° C. for 20 seconds, and extension
reaction at 68° C. for 1 minute. After purification and
confirmation of the length of the PCR product in 2% agarose gel
electrophoresis, the resulting scFv genes were subjected to DNA
sequencing. DNA sequences of scFvs were determined using a 3730 DNA
analyzer (Applied Biosystem, Foster City, Calif.).

Binding Assays on TLC Plates

[0127]400 pmol of artificial glycolipids dissolved in
chloroform/methanol/water (50:55:18, v/v) were dot-blotted onto TLC
plates. Alternatively, mixtures containing 1 μg each of artificial
glycolipids were spotted on aluminum-backed silica gel 60
high-performance TLC plates (10 cm-length; Merck, Darmstadt, Germany),
and were developed with a solvent system of chloroform/methanol/water
(60:35:8, v/v). After drying, these plates were soaked for 30 seconds in
n-hexane containing 0.1% (w/v) Plexigum P28 (Sigma-Aldrich) and then
blocked with 3% BSA/TBS at room temperature for 1 hour followed by
washing with TBS. The plates were then overlaid with phage antibody
(1013 cfu/ml) at 4° C. overnight followed by overlay with
mouse anti-M13 phage coat protein (p8) IgG (5000-fold dilution) at room
temperature for 1 hour. The binding of phage antibodies to artificial
glycolipids was detected by a combination of HRP-labeled anti-mouse IgG
(MBL, Nagoya, Japan) and a chemiluminescent reagent (ECL® Western
blotting detection reagents, Amersham Bio-Sciences, Buckinghamshire, UK)
according to the manufacturer's instructions.

Determination of Carbohydrate Specificity of Phage Antibodies Through
Examination of Their Reactivity to Natural Glycoproteins

[0128]Fetuin, asialo-fetuin, RNase A, and RNase B (5 μg/lane) were
separated by SDS-polyacrylamide gel electrophoresis (10% gel) and blotted
onto a PVDF membrane. The SDS-PAGE gel was stained with Coomassie
Brilliant Blue. The reactivity of phage antibody to natural glycoproteins
was examined by Western blotting with M3-7 phage antibody followed by
HRP-labeled anti-M13 phage antibody as described above.

[0130]scFv proteins were produced by the following method for use in the
specificity assay to various BSA-conjugated oligosaccharides. TOP10F'-FS2
cells were infected with Man3-specific phages, and cultured in 5 ml of
SBS with 1% glucose, 50 μg/ml carbenicillin, and 50 μg/ml
spectinomycin at 25° C. for 16 hours. Next, the infected cells
were collected by centrifugation, then suspended, and cultured in 40 ml
of SBS with carbenicillin and spectinomycin at 30° C. for 3 hours.
After 1 mM IPTG was added, the suspension was cultured at 30° C.
for 16 hours to induce scFv proteins, and the supernatant was collected.
After dot-blotting onto a nitrocellulose membrane, HRP-labeled anti-E-tag
antibody was added to confirm the expression of scFv proteins.

[0131]The ELISA plate was coated with 50 μl of BSA-conjugated
oligosaccharide (10 μl/ml) at 4° C. for 16 hours. The
supernatant of the culture solution was added to the wells (50
μl/well) and left at 37° C. for 2 hour. Binding of scFv
proteins to oligosaccharides was assayed according to the above method
with use of HRP-labeled anti-E-tag antibody.

[0132]SPR analyses were performed at 25° C. Solutions were freshly
prepared, degassed, and filtered through 0.22-μm pore filter. Binding
property of phage antibodies were determined by SPR using BIAcore X
(Pharmacia Biosensor). Used in this analysis were HPA sensor chips where
aliphatic chains were covalently bound to a gold surface, and 10 mM HEPES
(pH 7.4) containing 150 mM NaCl (HBS) as a running buffer. Immobilization
of artificial glycolipids on the HPA sensor chip surface was carried out
according to the manufacturer's protocol. Briefly, a lipid monolayer with
carbohydrates on the surface was formed on the HPA sensor chip by adding
artificial glycolipids bearing liposomes. Before immobilization, the
surface was cleaned by injection of nonionic detergent, 40 mM MEGA9
(Dojindo, Kumamoto, Japan) and 50% ethanol at a flow rate of 5 μl/min.
DPPC or Man3-DPPE/DPPC (1:10, mol/mol) was then applied to the sensor
chip surface at a low flow rate of 2 μl/min. To remove multilamellar
structures from the lipid surface, 5 μl of 10 mM NaOH was injected at
a flow rate of 5 μl/min. Phage antibody samples that had been dialyzed
against HBS were allowed to flow for 10 minutes over the surface of the
chips at a flow rate of 5 μl/min. To examine the competitive
inhibitory effect on SPR, phage antibody samples were mixed with 10 mM or
100 mM D-mannose or α-methyl-D-mannopyranoside (Nacalai Tesque,
Kyoto, Japan), and then incubated at 4° C. for 1 hour.

[0133]SPR analyses of scFv protein samples were performed at a flow rate
of 5 μl/min with BIAcore 3000 using the Sensor chip HPA or the Sensor
chip SA (Streptavidin). Immobilization of DPPC, Man3-DPPE/DPPC (1:10,
mol/mol), Man2-DPPE/DPPC (1:10, mol/mol), Man5-DPPE/DPPC (1:10, mol/mol)
to HPA sensor chips was performed as described above. The amounts of
immobilized ligands were calculated from the molar ratio of the DPPE
conjugates included in the lipid layer, which indicated that DPPC,
Man2-DPPE, Man3-DPPE, and Man5-DPPE were 0.43 pmol, 0.31 pmol, 0.48 pmol,
and 0.30 pmol, respectively. HBS containing 0.01% Tween 20 (HBS-T) was
used as a running buffer when SA sensor chips were used. SA sensor chip
surface was activated with three consecutive 1-min injections of
activating buffer (1 M NaCl containing 50 mM NaOH) prior to
immobilization of biotinylated ligands. Glucose-sp-Biotin,
Man3-sp-Biotin, and Lewis b-sp-Biotin (0.1 mM) were diluted with HBS-T to
0.05 μM, 10 μl of which were injected manually at a flow rate of 1
μl/min until RU of 100-150 is achieved. Unlike artificial glycolipids
used above, these ligands contained a spacer (sp) consisting of a
structure of --O(CH2)3NHCO(CH2)5NH--. The amounts of
immobilized biotinylated carbohydrates were estimated to be 0.25 pmol,
0.23 pmol, and 0.13 pmol, respectively. The scFv samples that had been
dialyzed against HBS-T were injected at a flow rate of 1 μl/min for 4
minutes over the surface of the chips. After one sample was assayed, the
sensor chip surfaces were regenerated by treating with 10 mM glycine-HCl
(pH 1.5) for 1 minute, followed by washing with a continuous flow of
HBS-T before the next sample was injected.

[0135]The phage displayed human scFv library was subjected to four rounds
of panning against Man3-DPPE. The structure of Man3-DPPE is presented in
FIG. 1B. Man3-DPPE (2 μg/well) was used for panning according to
abovementioned methods. Of 672 clones screened by ELISA using Man3-DPPE
(1 μg/well) as an antigen, 40 or more clones was ELISA-positive with
S/N of >2. Of those, 25 positive clones encoding scFvs were selected
as candidates for phage antibodies directed against the Man3 structure.
DNA sequencing of scFv regions of those phage clones revealed that all
the clones analyzed had different sequences. Characteristics of 12 clones
are shown in Table 1.

[0136]In order to quickly identify which phage antibodies have specificity
and high affinity for the antigen used, it is necessary to establish
methods suitable for characterizing scFv-presenting phages as phage
antibodies. Although expression, isolation, and characterization of scFv
proteins are ultimately required, expression and isolation of scFv
proteins are labor-intensive and time-consuming, and yet primary
information on phage clones is essential to select candidate clones for
further characterization. For this purpose, a set of artificial
glycolipids was individually spotted on TLC plates (FIG. 2A), and then
phage antibodies were overlaid on the plates to allow their binding to
the artificial glycolipids. These experiments revealed that phage
antibodies showed specificity to Man3, as well as to Man2 and Man5 to a
lesser extent as compared to Man3 (FIG. 2A). In addition, since binding
to GN2Man3 and DPPE was not observed, it was also demonstrated that the
phage antibodies examined bound to non-reducing mannose residues but not
to the lipid portion of artificial glycolipids. The structures of
artificial glycolipids used in these experiments are illustrated in FIG.
2B.

(3) Binding of Phage Antibodies to Natural Glycoproteins

[0137]Phage antibodies which were isolated by using Man3-DPPE as a target
antigen were shown to have specificity to non-reducing mannose residues
of artificial glycolipids. Therefore, it was next examined whether these
phage antibodies can bind to the non-reducing mannose residues of
glycoproteins. It was clearly shown that M3-7 phage antibody bound to
RNase B carrying high mannose type oligosaccharides but not to
non-glycosylated RNase A or to fetuin carrying complex type and O-linked
oligosaccharides (FIG. 3).

(4) SPR Analyses of Man3-Specific scFv-Displaying Phage Antibodies

[0138]After specificity of phage antibodies to Man3 was confirmed by ELISA
and TLC-overlay assays, SPR was used to observe the binding kinetics of
phage antibodies. A sensorgram showing the binding of M3-7 phage to
Man3-DPPE (the sensorgram was obtained by subtracting the binding of
phage antibody to Man3-DPPE by that of DPPC alone) confirmed the
Man3-specific binding property of the examined phage antibody (FIG. 4;
sensorgram A). This result was further supported by the fact that (i) the
binding of phage antibody was competitively inhibited by mannose
(sensorgram C) or α-methyl-D-mannoside, and (ii) a control phage
antibody which displayed human IGF-I receptor specific scFv did not bind
to Man3-DPPE (sensorgram B).

(5) Expression and Characterization of Man3-Specific scFvs as Soluble
Proteins

[0139]scFv proteins were expressed as soluble proteins for further
analysis with regard to their carbohydrate specificity. The content of
scFv proteins was compared among supernatants, periplasmic fractions, and
whole cell extracts by SDS-PAGE and subsequent immunoblotting with
anti-E-tag antibody. As a result, it was revealed that whole cell extract
fractions contained the most scFv proteins expressed. The whole cell
fractions were thus used for ELISA and SPR analyses (FIG. 5). Although
the fractions used were unpurified (FIG. 5 Left), they contained E-tag
reactive scFv proteins (FIG. 5 Right). The amounts of scFv proteins
expressed by E. coli cells that had been infected with phages under the
same condition to that of this study can also be considered to be within
a similar range (FIG. 5 Right). The binding abilities of scFv proteins
M3-7 and M3-8 to mannose residues were confirmed by ELISA with use of
synthetic glycoproteins (FIG. 6).

(6) SPR Analysis of Man3-Specific scFvs as Soluble Proteins

[0140]scFv protein samples (FIG. 5) were used to analyze the binding
kinetics of Man3-specific scFv proteins by SPR using immobilized
artificial glycolipids (FIG. 7). As a result, the scFv protein derived
from the M3-7 phage clone showed similar relative kinetic of binding to
Man3-DPPE and Man5-DPPE, which were higher than the binding kinetic to
DPPE. On the other hand, the binding kinetic to Man2-DPPE was lower than
the binding kinetic to DPPE (FIG. 7).

[0141]SPR Analyses of biotinylated carbohydrates were also carried out. In
view of the binding kinetics of scFv protein samples to Man3-sp-biotin,
Lewis b-sp-biotin, and glucose-sp-biotin, the scFv protein M3-7 bound to
Man3 at much higher affinity than to Lewis b which is structurally
irrelevant (the affinity of the scFv protein M3-7 to Lewis b was very low
but significant) (FIG. 8). On the other hand, these scFv proteins did not
appear to bind to glucose used as a control. The sensorgram of anti-FLAG
scFv protein should be regarded as a control.

[0142]The sensorgrams of the bindings of scFv proteins to Man3-DPPE (FIG.
7) and Man3-biotin (FIG. 8) were respectively subtracted by the
sensorgrams on DPPE (FIG. 9A) and glucose-biotin (FIG. 9B). From these
two types of results, the specific binding to Man3 was compared (FIG. 9).
These data show several facts regarding relative affinities between scFv
proteins examined. For example, the scFv protein obtained from M3-7 has
an affinity to Man3 structure.

INDUSTRIAL APPLICABILITY

[0143]The present invention provides antibodies which recognize
non-reducing mannose residues, and nucleic acids encoding these
antibodies. The antibodies of the present invention are able to bind,
with specificity and high affinity, to various carbohydrates including
carbohydrate antigens, individual recognition of which is not feasible.
Further, gene manipulation of single chain antibodies of the present
invention enables production of modified single chain antibodies, and
mass production of single chain antibodies with ease. In addition, gene
manipulation of the antibodies obtained by the present invention enables
carbohydrate-specific delivery of proteins such as toxins and enzymes.

[0146]FIG. 3 shows binding of phage antibodies to natural glycoproteins.
Reactivity of M3-7 phage antibody to natural glycoproteins (fetuin,
asialofetuin, RNase A, and RNase B) was examined by Western blotting. The
protein bands were stained with Coomassie Brilliant Blue. Western
blotting with M3-7 phage antibody and HRP-labeled anti-M13 phage antibody
was carried out to identify the band where the phage antibodies was
bound.

[0147]FIG. 4 shows SPR analysis of Man3-specific scFv-displaying phage
antibodies. M3-7 phage antibody (A) was subjected to SPR analysis with
Man3-DPPE. Anti-IGF-I receptor scFv-phage was used as a negative control
(B). A competitive inhibition test was performed with M3-7 phage antibody
in the presence of 100 mM mannose (C). The sensorgrams shown are specific
binding to Man3, which were obtained by subtracting the sensorgrams on
DPPC from those obtained on specific Man3-DPPE.

[0148]FIG. 5 shows expression of Man3-specific scFv as a soluble protein.
Left: SDS-PAGE of scFv protein (M3-7) expressed in E. coli TOP10F' as
well as anti-FLAG scFv protein as a control was carried out. The gel was
stained with Coomassie Brilliant Blue. Right: Western blotting of scFv
proteins with an anti-E-tag antibody was performed.

[0151]FIG. 8 shows SPR analysis of Man3-specific scFv proteins on chip
immobilized with Man3-sp-biotin, Lewis b-sp-biotin, or glucose-sp-biotin.
The thick line indicates Man3-sp-biotin; the dotted line indicates Lewis
b-sp-biotin; and the thin line indicates glucose-sp-biotin.

[0152]FIG. 9 shows comparison of the bindings of scFv proteins to Man3
moiety. The comparison was made between two types of sensorgrams of scFv
proteins with respect to Man3-DPPE (FIG. 7) and Man3-biotin (FIG. 8).
Specific bindings were obtained by subtracting the sensorgrams on DPPE
(FIG. 9A) or glucose-biotin (FIG. 9B) from the respective sensorgrams.